Capture methodologies for circulating cell free dna

ABSTRACT

A nucleic acid patch method for amplifying target nucleic acid sequences in circulating free DNA or residual DNA samples where the defining ends of the target nucleic acid sequences are unknown.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation of U.S. Utility application Ser. No.15/969,525, filed May 2, 2018, which is a Continuation of U.S. Utilityapplication Ser. No. 13/794,267, filed on Mar. 11, 2013. The entiredisclosure of all the above documents is herein incorporated byreference.

BACKGROUND 1. Field of the Invention

This disclosure is related to the field of devices, methods, systems andprocesses for capturing and amplifying targeted regions on circulatingcell free DNA fragments. Specifically, for capturing and amplifyingtargeted regions on genomic DNA where the end points of the desiredtarget are unknown or a portion of the end points of the desired targetare known but it is unknown how much of the end point is present.

2. Description of Related Art

The completion of the decoding of the canonical genome sequences of allmajor model organisms, as well as the human species, has thrown open thedoor to elucidating the candidate genes associated with various humandiseases. The application of the genetic origins of human disease can bevery powerful to the understanding and development of treatments forthese diseases. Examples of successful application of the genetic basisof disease in the clinic and clinical research settings includes thesequencing of candidate disease loci in targeted populations, such asthe Ashkenazi Jews (Weinstein 2007), the sequencing of variants in drugmetabolism genes to adjust dosage (Marsh and McLeod 2006), and theidentification of genetic defects in cancer that make tumors moreresponsive to certain types of treatments (Marsh and McLeod 2006).Accordingly, medical re-sequencing of candidate genes in individualsamples is becoming increasingly important in clinical settings and inclinical research. Medical re-sequencing requires the amplification andsequencing of many candidate genes in many patient samples. However, theability to fully embrace the promise of the clinical-application ofgenetic-based research necessitates the development of new technology tolower the cost and increase the throughput of medical re-sequencing tomake clinical applications more feasible.

As noted in United States Patent Application Publication No.:2010/0129874, the entirety of which is specifically incorporated hereinby reference to the extent not inconsistent with the disclosures of thispatent, many of the current methods for analyzing sequence variation ina subset of the human genome generally rely on polymerase chain reaction(“PCR”) to amplify targeted sequences (Greenman, et al. 2007; Sjoblom,et al., 2006; Wood, et al., 2007). However, efforts to multiplex PCR(i.e., target many regions across multiple samples in a single process)have been hampered by a dramatic increase in mispriming events as moreprimer pairs are used (Fan, et al. 2006). Further, the larger number ofprimer pairs utilized in multiplex PCR often results in inter-primerinteractions that prevent amplification (Han, et al.). Therefore,separate PCRs for each region of interest generally must be performed.(Greenman, et al., 2007; Sjoblom, et al., 2006; Wood, et al., 2007).This creates a costly approach when hundreds of individual PCRs must beperformed for each sample. Further, these methods often have inherentproblems with multiplicity (i.e., the number of independent capturereactions which can be performed simultaneously in a single reaction),specificity (i.e., measured as the fraction of captured nucleic acidsthat derive from targeted regions), and uniformity (i.e., relativeabundance of targeted sequences after selective capture). Ideally, amultiplex PCR model would perform each of these performance parameters(multiplicity, specificity and uniformity) well. As noted in UnitedStates Patent Application Publication No.: 2010/0129874, this was notaccomplished by the currently utilized systems. Another problem was thatcurrently utilized multiplex PCR methods and systems required a largeamount of starting DNA to supply enough template for all of the requiredindividual PCR reactions. This was a problem since DNA can serve as alimiting factor when working with clinical samples.

Because of these problems, there was a need in the art for a multiplexedPCR method that simultaneously amplified many targeted regions from asmall amount of nucleic acid. United States Patent ApplicationPublication No.: 2010/0129874 disclosed a method for amplifying at leasttwo different nucleic acid sequences utilizing a multiplexed nucleicacid patch PCR which, in part, responded to this need in the art.

In general, the method disclosed in United States Patent ApplicationPublication No.: 2010/0129874 relies on two rounds of target-specificenrichment, with discrete clean-up steps between each round, to confermore specific targeting and amplification than the previously known PCRsystems and methodologies. Specifically, the disclosed methods requirefour oligonucleotide hybridizations per locus, resulting in morespecific amplification than standard multiplex PCR, which requires onlytwo hybridizations per locus.

In the first round, targeting primer pairs are designed for each targetregion (i.e., specific regions of interest within genomic DNA), and alow number of PCR cycles are performed. This low cycle amplificationserves two functions: 1) it defines the target regions; and 2) itdifferentiates the target regions from non-targeted background DNA. Theprimers utilized in this round are designed to include uracil instead ofthiamine, and are cleaved and removed by enzymes following the initialamplification. At the end of the first round, the ends of the targetregion are now internal to the PCR primer sequences.

In the second round, a target-specific enrichment, “patcholigonucleotides,” are employed. The patch oligonucleotides arecomprised of a string of oligonucleotides of variable length thatcontain, at a minimum, a sequence that is the reverse compliment to atleast a portion of a sequence that defines the targeted region. Stateddifferently, each patch oligonucleotide is designed specifically for theends of each target region, slowing ligation of universal adapters, anda protecting group. The patch oligonucleotides are annealed to thetargeted regions and serve as a patch between targeted amplicons anduniversal primers. This targeting step delivers a higher level ofspecificity as only targeted regions can anneal with patcholigonucleotides. The universal primers, which anneal to the universalregion of the patch oligonucleotides, then ligate to each targetamplicon. This reaction is highly specific because thermostable ligasesare sensitive to mismatched bases near the ligation junction (Barany1991). An added level of selectivity is gained by degrading misprimingproducts as well as the genomic DNA with exonuclease. The selectedamplicons are protected from degradation by a 3′ modification on theuniversal primer. This hybridization and ligation. of patcholigonucleotides to primer-depleted amplicons is followed bymulti-template PCR amplification with primers corresponding to theuniversal sequences.

In the second round, the patch oligonucleotides confer additional andvery high specificity in targeting regions of interest because theligation is dependent on sequences immediately internal to the originalprimers used in the initial low cycle PCR. Thus, a further level ofspecificity is achieved by degrading any misprimed product and genomicDNA. Stated differently, enzymatic digestion is utilized to remove allnon-protected DNA including any misprimed segment from the initiallimited cycle step. The cleanup ensures that only the targeted regionsare loaded onto the next generation sequencer for universalamplification—all off-target amplicons are degraded. Thus, in thisprocess, all of the targeted regions are amplified and enrichedsimultaneously, in one tube, start to finish.

In sum, the methods disclosed in United States Patent ApplicationPublication No.: 2010/0129874 addressed the need in the art for amultiplexed PCR method with the ability to amplify many targeted regionsfrom a small amount of nucleic acid, allowing for the targeting of manyregions across multiple samples, thereby providing an effective solutionto maximize throughput capacity of sequencers.

While an advance in the art, the methods disclosed in United StatesPatent Application Publication No.: 2010/0129874 are still limited.Because these methods are based on defining the ends of nucleic acidsequences, they are generally not applicable in situations where thetargeted region is located in fragmented DNA, residual DNA orcirculating cell free DNA (circulating cell free DNA is produced throughthe process of cellular apoptosis and released into circulation). Stateddifferently, the methods disclosed in United States Patent ApplicationPublication No.: 2010/0129874 are only applicable if the end of at leasttwo nucleic acid sequences to which patches can be annealed are known.Thus, these methods are not applicable to capturing DNA fragments andcirculating cell free DNA—i.e., situations in which the defining ends ofthe nucleic acid target sequences are unknown. Amongst otherapplications, capturing DNA fragments and circulating cell free DNA isimportant to the identification of genetic defects in fetal DNAcirculating in maternal blood for the diagnosis of prenatal healthissues. See Lo Y M, et al., “Presence of fetal DNA in maternal plasmaand serum,” Lancet, 350(9076):485-7 (Aug. 16, 1997); Palomaki G E, etal., “DNA Sequencing of Maternal Plasma to Detect Down syndrome: AnInternational Clinical Validation,” Genet Med, Vol 13:No 11 (November2011).

SUMMARY

Because of these and other problems in the art, described herein, amongother things, is a nucleic acid patch method for amplifying targetnucleic acid sequences in circulating free DNA or residual DNA. sampleswhere the defining ends of the target nucleic acid sequences areunknown.

In an embodiment, there is described a method for the capture andamplification of a targeted region of fragmented DNA (fgDNA), the methodcomprising: choosing one or more targeted regions of fgDNA foramplification; adding oligonucleotide patches to the fgDNA, theoligonucleotide patches binding to the ends of the one or more targetedregions creating double stranded DNA at the bound ends of the one ormore targeted regions; adding enzymes to the fgDNA, the enzymes cleavingto the single-stranded DNA, resulting in a product with blunted doublestranded DNA defined by the oligonucleotide patches bound to one or morereduced targeted regions; performing polymerase chain reaction; andsequencing the polymerase chain reaction amplicons of the one or moretargeted regions.

In an embodiment of the method, the oligonucleotide patches contain oneor more nucleotide terminal sequences.

In an embodiment of the method, the nucleotide terminal sequencescomprise an adenine and a universal primer sequence.

In an embodiment of the method, the oligonucleotide patches arecomprised of universal primers, protecting groups and nucleotideterminal sequences.

In an embodiment of the method, a multiplicity of oligonucleotide patchpairs are added to the reaction concurrently.

There is also described herein, a method for the capture andamplification of a targeted region of fragmented DNA, the methodcomprising: choosing one or more minimal targeted regions of fgDNA forcapture and amplification; adding oligonucleotide patches to the fgDNA,the oligonucleotide patches comprising a universal primer sequence,protecting groups on at least on end, and a string of oligonucleotidesof variable length, the string containing a sequence that is the reversecompliment to an end sequence that defines the one or more minimaltargeted regions of fgDNA; binding the reverse nucleotide portion of theoligonucleotide patches to a portion of the one or more minimal targetedregions; filling in a remaining single-stranded region between theuniversal primer sequences and the oligonucleotide patches withpolymerase; using ligation to join the universal primer sequences andthe fgDNA into a single molecule; removing all unprotected primers andnon-targeted DNA with enzymes; performing polymerase chain reaction; andsequencing the products of the polymerase chain reaction.

In an embodiment of the method, a multiplicity of oligonucleotidepatches are added concurrently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a step of an embodiment of the patch digest protectionprocess in which the nucleic acid sequence of the oligonucleotidepatches binds to targeted region(s) of fragmented DNA to create doublestranded DNA at the bound ends of the targeted region(s).

FIG. 2 depicts a step of an embodiment of the patch digestion process inwhich enzymes are utilized to cleave to the single-stranded DNA on theouter flanks of the oligonucleotide patches, resulting in a product withblunted double-stranded DNA ends defined by patches.

FIG. 3 depicts a step of an embodiment of the patch digestion process inwhich the oligonucleotide patches bound to the targeted regions serve asa connector to allow the ligation of the universal primers containingprotecting groups to the end of the reduced targeted region.

FIG. 4 depicts a step of an embodiment of the patch digestion process inwhich polymerase chain reaction is performed using universal primers tosimultaneously amplify multiple targeted regions in the same reaction.

FIG. 5 depicts an embodiment of the patch the gap process.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

By way of background, polymerase chain reaction (“PCR”) amplifiesspecific nucleic acid sequences through a series of manipulationsincluding denaturation, annealing of oligonucleotide primer pairs andextension of the primers with DNA polymerase. These steps can besequentially repeated, resulting in an exponential amplification of thenumber of copies of the original target sequence. Multiplex PCR is avariation of PCR that enables the simultaneous amplification of manytargets of interest in one reaction by using more than one pair ofprimers. As noted previously, current multiplex PCR methods are hamperedby the amplification of mispriming events, inter-primer interactionsthat prevent amplification as more primer pairs are used and cost.

Disclosed herein are various methods, processes and systems for theamplification of targeted sequences in fragmented genomic DNA where theends abutting the targeted genomic sequences are unknown. In general, atargeted region is a user defined region to be captured for sequencinglarge portions of genomic DNA. It is often unknown what exact sequenceis joining the two regions that are linked together.

In one embodiment, the method, process and system for the capture andamplification of a targeted region of fragmented DNA is patch digestprotection (PDP). In general, in PDP one or more targeted regions offgDNA (i.e., genomic DNA (gDNA) fragmented into a distribution of sizes)are chosen for capture and amplification by the PDP process. In a firststep of PDP, oligonucleotide patches are added to the fgDNA.Oligonucleotide patches are a string of oligonucleotides of variablelength that contain, at a minimum, a sequence that is the reversecompliment to an end sequence that defines the targeted region of fgDNA.It is contemplated that the oligonucleotide patches disclosed hereinwill also contain one or more nucleotide terminal sequences, preferablyan adenine and a universal primer sequence, and protecting groups on oneor both ends. Among other functions, the terminal sequences prevent thepolymerase extension of the patch oligonucleotides. Theseoligonucleotide patches will be discussed more fully later in thisapplication. In certain embodiments, it is contemplated that amultiplicity of oligonucleotide patch pairs (AM) are added to thereaction concurrently. Once introduced, the oligonucleotide patches bindto the ends of the targeted region(s), protecting groups on one or bothends. This is depicted in FIG. 1 where patch 1A and patch 1B bind to theends of the targeted region. This binding of the nucleic acid sequenceof the patches to the targeted region creates a double stranded DNA atthe bound ends of the targeted region, as depicted in FIG. 1.

In a second step of the PDP process, following binding of theoligonucleotide patches to the ends of the targeted region, enzymes areutilized to cleave to the single-stranded DNA on the outer flanks of theoligonucleotide patches. These enzymes will be discussed more fullylater in this application. This second step, which is depicted in FIG.2, results in a product with blunted double stranded DNA ends defined bythe patches (patches 1A and 1B in the depicted Figure) are bound to areduced targeted region. The reduced target regions are protected fromdegradation in this step because they are flanked by double-strandedDNA. In general, as the term is used herein, a reduced targeted regionis the subsection of fgDNA equal to or larger than a targeted regions,whose ends are defined by patches.

As noted previously, the oligonucleotide patches are comprised ofuniversal primers, in addition to protecting groups and a nucleic acidsequence that will bind to a targeted region. Through these universalprimers, the patches, in addition to binding to the target regionthereby isolating the targeted region, serve as a connector to allow theligation of the universal primers containing protecting groups to theend of the reduced targeted region. This process is depicted in FIG. 3.In a final step, as depicted in FIG. 4, PCR is performed using universalprimers to simultaneously amplify multiple targeted regions in the samereaction. After amplification, the PCR product amplicons of the targetedregions can be sequenced.

In another embodiment, the method, process and system for the captureand amplification of a targeted region of fragmented DNA is patch thegap (PtG). In one contemplated scenario, patch the gap is utilized whenit is known what the end of the targeted region is, but it is unknownhow much is there. In PtG, one or more minimal targeted regions of fgDNAare chosen for capture and amplification by the PtG process. As the termis used herein, minimal targeted regions are fgDNA segments that containa targeted region with ends that cannot be precisely defined by patches.In a first step of PtG, oligonucleotide patches are added to the fgDNA.The oligonucleotide patches utilized in PtG are comprised of a string ofoligonucleotides of variable length that contain, at a minimum, asequence that is the reverse compliment to an end sequence that definesthe targeted region of fgDNA. Further, it is contemplated that theoligonucleotide patches disclosed herein for use in fgDNA also contain auniversal primer sequence (UPR) and protecting groups on one or bothends. These oligonucleotide patches will be discussed more fully laterin this application. In certain embodiments, it is contemplated that amultiplicity of patch pairs (A/B) are added to the reactionconcurrently. Once added to the fgDNA, the reverse nucleotide portion ofthe patches anneal (or bind) to a portion of the minimal targeted regionof the fgDNA, the universal primer sequences and the universal primer,as depicted in FIG. 5.

In a next step, the remaining single-stranded region between the UPR andthe patches is filled in by polymerase as depicted in FIG. 5. Asdepicted, the polymerase extension on the 5′ end of the fgDNA isinitiated from the universal primer and polymerizes double-stranded DNAuntil it reaches the fgDNA. Conversely, the polymerase extension on the3′ end is initiated from the fgDNA and polymerizes double stranded DNAuntil it reaches the end of the universal primer. Generally, apolymerase that lacks both strand displacement and 5′-3′ exonucleaseactivity is utilized so that pocessivity of the polymerase is stoppedwhen it reaches double-stranded DNA.

In a third step in PtG, ligation is used to join the universal primersand fgDNA into a single molecule. As noted previously, the universalprimers generally contain protecting groups. In a next step, enzymes areused to remove all of the unprotected primers and non-targeted DNA. In afinal step, PCR is performed using universal primers to simultaneouslyamplify many targeted regions in the same reaction. Subsequent to thisamplification, the PCR products can be sequenced.

In order to better understand these systems, methods and processes ofPDP and PtG disclosed herein, the following terms, components andconditions of the described processes and methods are further defined.

A) Nucleic Acid Template

The methods, systems and processes disclosed herein may be used toamplify nucleic acid sequences. Generally, nucleic acid sequences arefound in a nucleic acid template. It should be understood that thenucleic acid template described herein may be from any sample thatcontains nucleic acid molecules. The nucleic acid template may be fromhumans, animals, plants, microorganisms, or viruses. The sample may befresh, from archeological or forensic samples, or from preserved samplessuch as paraffin-embedded tissue. The sample may be a solid tissue or aphysiological fluid such as blood, serum, plasma, saliva, ocular lensfluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid,lymphatic fluid, mucous, synovial fluid, peritoneal fluid, sputum fluidor amniotic fluid. Nucleic acid templates may be prepared from thesample methods well known to those of ordinary skill in the art.Alternatively, the sample containing the nucleic acid template may beused directly.

The nucleic acid template may be DNA, RNA, or a complementary DNA (cDNA)sequence that is synthesized from a mature messenger RNA. If the nucleicacid template is RNA, the RNA may be reverse transcribed to DNA usingmethods well known to persons skilled in the art. In a preferredembodiment, the nucleic acid template is DNA.

In some embodiments, suitable quantities of nucleic acid template forthe invention may be 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001 .mu.g or less. In preferred embodiments, suitablequantities of nucleic acid template for the invention may be 1000, 900,675, 450, 225, 112, 70, 50, 20, 1.6 ng or less.

In some embodiments, the nucleic acid template may be treated to preparethe template for specific applications of the invention. In oneembodiment, the nucleic acid template may be treated with bisulfite todetermine the pattern of methylation. Nucleic acid templates may betreated with bisulfite using methods well known to those of skill in theart, and may be performed using commercially available reagents,following manufacturer's protocols, such as by using the EZ DNAMethylation-Gold Kit™ (Zymo Research), the Imprint™ DNA Modification Kit(Sigma), or the like.

Further, in certain preferred embodiments, the method and processes ofamplification. discussed herein are used to amplify nucleic acidsequences in circulating cell free DNA, DNA fragments and/or residualDNA. Examples of such circulating cell free DNA amplificationapplications include, but are not limited to, detecting geneticdisorders in a fetus using blood samples from a pregnant woman;diagnosing and monitoring cancer patients for cancer type, stage,recurrence and/or drug resistance; discovery, detection, and monitoringof biomarkers in other disease states, such as cardiac disease,neurological disorders, or infectious disease; detecting geneticmaterial from microbes and pathogens in environmental, medical orindustrial samples;

and determining the identity of an individual/organism from a residualsample, for example, a fingerprint of a suspect from a crime scene or agenetically modified corn strain in a GMO-free cereal (in both of thesescenarios, there is only a small amount of DNA material).

B) Polymerase

In one embodiment, the nucleotide polymerase disclosed herein andutilized, for example, in the disclosed PtG process, may be a DNApolymerase. In another embodiment, the nucleotide polymerase may be athermostable polymerase. A thermostable polymerase is an enzyme that isrelatively stable to heat and eliminates the need to add enzyme prior toeach PCR cycle. Non-limiting examples of thermostable polymerases mayinclude polymerases isolated from the thermophilic bacteria Thermusaquaticus (Taq polymerase), Thermus thermophilus (Tth polymerase),Thermococcus litoralis (Tli or VENT™ polymerase), Pyrococcus furiosus(Pfu or DEEPVENT™ polymerase), Pyrococcus woosii (Pwo polymerase) andother Pyrococcus species, Bacillus stearothermophilus (Bst polymerase),Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum(Tac polymerase), Thermus rubber (Tru polymerase), Thermus brockianus(DYNAZYME™ polymerase) Thermotoga neapolitana (Tne polymerase),Thermotoga maritime (Tma) and other species of the Thermotoga genus (Tsppolymerase), and Methanobacterium thermoautotrophicum (Mth polymerase).The applicable methods and processes disclosed herein may contain morethan one thermostable polymerase enzyme with complementary propertiesleading to more efficient amplification of target sequences. Forexample, a nucleotide polymerase with high processivity (the ability tocopy large nucleotide segments) may be complemented with anothernucleotide polymerase with proofreading capabilities (the ability tocorrect mistakes during elongation of target nucleic acid sequence),thus creating a PCR reaction that can copy a long target sequence withhigh fidelity. The thermostable polymerase may be used in its wild typeform. Alternatively, the polymerase may be modified to contain afragment of the enzyme or to contain a mutation that provides beneficialproperties to facilitate the PCR reaction. In one embodiment, thethermostable polymerase may be Taq polymerase. Many variants of Taqpolymerase with enhanced properties are known and include AmpliTaq™,AmpliTaq™ Stoffel fragment, SuperTaq™, SuperTaq™ plus, LA Taq™, LAproTaq™, and EX Taq™.

C) PCR Reaction Conditions

Buffer conditions for PCR reactions are known to those of ordinary skillin the art. PCR buffers may generally contain about 10-50 mM Tris-HCl pH8.3, up to about 70 mM KCl, about 1.5 mM or higher MgCl.sub.2, to about50-200 .mu.M each of dATP, dCTP, dGTP and dTTP, gelatin or BSA to about100 .mu.g/ml, and/or non-ionic detergents such as Tween-20 or NonidetP-40 or Triton X-100 at about 0.05-0.10% v/v. In some embodiments,betaine may be added to the PCR reactions at about 0.25 to about 1 M.

In some embodiments, the multiplex PCR reaction performed subsequent toPDP or PtG may contain 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100, 1200 or more primer pairs. Not allprimer pairs will amplify targets with the same efficiency. In someembodiments, PCR primer pairs with similar amplification efficiency maybe pooled in separate multiplex PCR reactions to have betterrepresentation of all targets. These PCR reactions may be combined afteramplification.

In other embodiments, PCR amplification may be performed at a uniformtemperature (isothermal PCR). Examples of isothermal PCR methods mayinclude the ramification amplifying method and the helicase-dependentamplification method. In a preferred embodiment of the invention, PCRamplification may be by thermal cycling between a high temperature tomelt the nucleic acid strands, a lower temperature to anneal the primersto the target nucleic acid, and an intermediate temperature compatiblewith the nucleic acid polymerase to elongate the nucleic acid sequence.In one embodiment, the melting temperatures may be about 85, 86, 87, 88,89, 90, 95, or 100 degrees Celsius. In a preferred embodiment, themelting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98degrees Celsius. In another embodiment, the annealing temperatures maybe 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 degrees Celsius or more. In apreferred embodiment, the annealing temperature may be 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, or 72degrees Celsius. In yet another embodiment, the elongation temperaturemay be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 degrees Celsius ormore. In a preferred embodiment, the elongation temperature may be 70,71, 72, 73, 74, 75, 80 degrees Celsius or more.

In certain embodiments, the PCR reaction may be incubated at the meltingtemperature for about 5 to about 60 seconds. In a preferred embodiment,the PCR reaction may be incubated at the melting temperature for about30 seconds. In some embodiments, the PCR reaction may be incubated atthe annealing temperature for about 5 to about 60 seconds. In apreferred embodiment, the PCR reaction may be incubated at the annealingtemperature for about 30 seconds. In some embodiments, the PCR reactionmay be incubated at the elongation temperature for about 1 to about 10minutes. in a preferred embodiment, the PCR reaction may be incubated atthe elongation temperature for about 6 minutes. In some embodiments, thePCR reaction is pre-incubated at the melting temperature for about 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 minutes before cycling between the melting,annealing and elongation temperatures. In a preferred embodiment, thePCR reaction may be pre-incubated at the melting temperature for about 2minutes.

In several embodiments, the PCR reactions may be cycled between theinciting, annealing and elongation temperatures 2, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55 or more times. In a preferred embodiment, the PCRreactions may he cycled between the melting, annealing and elongationtemperatures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or moretimes.

D) Trimming Applications

It is contemplated in some embodiments of the process, methods andsystems disclosed herein, that the single-stranded DNA on the outerflanks of the patch oligonucleotides may be trimmed, leaving the reducedtargeted region defined by the double-stranded DNA created when theoligonucleotides bind to the targeted region. This, for example, occursin one step of the disclosed PDP process. in preferred embodiments, theunincorporated single-stranded DNA on the outer flanks of the patcholigonucleotides may be removed using enzymes such as exonucleases,enzymes that cleave nucleotides from the end of a polynucleotidesequence. In general, the reduced targeted regions in this process areprotected from degradation because they are flanked by double-strandedPatch-bound DNA. Enzymes may also be utilized in the process, method andsystems disclosed herein to remove all of the unprotected primers andnon-targeted DNA after the universal primers are ligated to the ends ofthe reduced targeted region.

E) Patches Oligonucleotides

In general, patch oligonucleotides, as that term is used herein, are astring of oligonucleotides of variable length that contain, at aminimum, a sequence that is the reverse complement to the sequence thatdefines a targeted region. Generally, in the process, methods andsystems disclosed herein, annealing of the restriction enzyme-directingoligonucleotides to the nucleic acid templates may be performed bymelting the nucleic acid strands at a high temperature, followed by alower temperature suitable for annealing the restrictionenzyme-directing oligonucleotides to target nucleic acid sequences. Inone embodiment, the melting temperatures may be about 85, 86, 87, 88,89, 90, 95, or 100 degrees Celsius. in a preferred embodiment, themelting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98degrees Celsius. In another embodiment, the annealing temperatures maybe about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 degrees Celsius or more.In a preferred embodiment, the annealing temperatures may be about 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 50, 51, or 52 degrees Celsius.

In other embodiments, the annealing reactions may be incubated at themelting temperature for about 5 to about 30 minutes. In a preferredembodiment, the annealing reactions may be incubated at the meltingtemperature for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 minutes. In some embodiments, the annealing reactions maybe incubated at the annealing temperature for about 1 to about 10minutes. In a preferred embodiment, the annealing reactions may beincubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 minutes.

F) Single Strand Specific Exonuclease Degradation

In the processes, methods and systems disclosed herein, single strandspecific exonuclease enzyme digestion of nucleic acid templatesprotected by locus-specific oligonucleotides patches may be used todefine ends of the targeted region. This is facilitated by upstream anddownstream oligonucleotide patches that anneal to the targeted regionand serve as protection against digestion by the single strand specificexonuclease enzymes. Thus, components of the exonuclease reaction mayinclude the nucleic acid sequence to be digested, one or more singlestrand specific exonuclease enzymes, the oligonucleotide patchesprotecting the nucleic acid targeted region, and salts and buffersessential for optimal activity of the exonucleases in the reaction.

Non-limiting examples of single strand specific exonuclease enzymessuitable for the methods of the invention may be exonuclease VII,exonuclease I, Red exonuclease, or Terminator™ 5′-Phosphate-DependentExonuclease (Epicentre Biotechnologies). The upstream and downstreamoligonucleotide patches may be designed using primer length, GC paircontent, and melting temperature criteria.

Annealing of the protecting oligonucleotides patches to the nucleic acidtargeted regions may generally be performed before addition of theexonuclease enzymes. In addition to the fgDNA, annealing reactions maygenerally contain about 1 pM to about 500 nM of each oligonucleotide. Insome embodiments, annealing of the oligonucleotides may be performed bymelting the nucleic acid strands at a high temperature, followed by alower temperature suitable for annealing the protecting oligonucleotidesto target loci. In one embodiment, the melting temperatures may be about85, 86, 87, 88, 89, 90, 95, or 100 degrees Celsius. In a preferredembodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95,96, 97 or 98 degrees Celsius. In another embodiment, the annealingtemperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 degreesCelsius or more. In a preferred embodiment, the annealing temperaturesmay be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, or 52 degrees Celsius.

In some embodiments, the annealing reactions may be incubated at themelting temperature for about 5 to about 30 minutes. In a preferredembodiment, the annealing reactions may be incubated at the meltingtemperature for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 minutes. In some embodiments, the annealing reactions maybe incubated at the annealing temperature for about 1 to about 10minutes. In a preferred embodiment, the annealing reactions may beincubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 minutes. After annealing of theprotecting oligonucleotides, the exonuclease enzymes may be added fordigestion.

G) Ligation of Universal Primer Sequences

One aspect of the PDP and PtG methods disclosed herein is the ligationof universal primer sequences to nucleic acid sequences. In general,this is facilitated by upstream and downstream nucleic acid patcholigonucleotides that anneal upstream and downstream of the targetnucleic acid sequences and serve as a patch between the desired sequenceand upstream and downstream universal primers to be ligated. Thus,nucleic acid patch ligation reactions generally contain the targetsequences, the upstream and downstream universal primers to be ligated,the upstream and downstream nucleic acid patch oligonucleotides to guidethe specific ligation of the universal primers, and the enzymes andother components needed for the ligation reaction.

1. Universal Primers

The upstream and downstream universal primers may be designed usingprimer length, GC pair content and melting temperature criteria. In someembodiments, the downstream universal primer may be modified tofacilitate further steps of the invention. In a specific embodiment, thedownstream universal primer may be modified with a 5′ phosphate group toenable ligation of the downstream universal primer to the amplicon. Inother specific embodiments, the 3′ end of the downstream universalprimer may be modified for protection against exonuclease digestion.Modifications at the 3′ end may be introduced at the time of synthesisor after synthesis through chemical means well known to those of skillin the art. Modifications may be 3′ terminal or slightly internal to the3′ end. Some examples of modifications that make nucleic acid sequencesexonuclease resistant include, but are not limited to, locked nucleicacids (LNA's), 3′-.linked amino groups, 3′ phosphorylation, the use of a3′-terminal cap (e.g., 3′-aminopropyl modification or by using a 3′-3′terminal linkage), phosphorothioate modifications, the use of attachmentchemistry or linker modification such as Digoxigenin NHS Ester,Cholesteryl-TEG, biotinylation, thiol modifications, or addition ofvarious fluorescent dyes and spacers such as C3 spacer. in a preferredembodiment, the downstream universal primer is protected fromexonuclease digestion by a C3 spacer.

2. Nucleic Acid Patch Primers

In some embodiments, an upstream and a downstream nucleic acid patcholigonucleotide may be designed for each targeted region of fgDNA. Insome preferred embodiments, the 5′ ends of the upstream nucleic acidpatch oligonucleotides may be complementary to sequences in the fgDNAand may be concatenated to upstream nucleotide sequences complementaryto the upstream universal primer sequence on the 3′ end. In otherpreferred embodiments, the 3′ ends of the downstream nucleic acid patcholigonucleotides may be complementary to downstream sequences in thefgDNA, and may be concatenated to nucleotide sequences complementary tothe downstream universal primer sequence on the 5′ end.

3. Ligation of Universal Primers

In some embodiments, the universal primers may be ligated to nucleicacid sequences. In a process similar to a PCR amplification reaction,multiple cycles of heating and cooling may be used to melt the targetnucleic acid sequence, anneal the nucleic acid patch and universalprimers, and ligate the universal primers to target nucleic acidsequences.

In some embodiments of the invention, the universal primers of theinvention may be ligated to the target nucleic acids using a DNA ligase.The ligase may be thermostable. In preferred embodiments, the ligase isa thermostable DNA ligase. A thermostable DNA ligase is an enzyme thatis relatively stable to heat and eliminates the need to add enzyme priorto each ligation cycle. Non-limiting examples of thermostable DNAligases may include Ampligase® Thermostable DNA Ligase, Taq DNA Ligasefrom Thermus aquaticus, Tfi DNA ligase from Thermus filiformis, Tth DNAligase from Thermus thermophilus, Thermo DNA ligase, Pfu DNA ligase fromPyrococcus furiosus, and thermostable DNA ligase from Aquifexpyrophilus. The thermostable polymerase may be used in its wild typeform, modified to contain a fragment of the enzyme, or to contain amutation that provides beneficial properties to facilitate the ligationreaction. In a preferred embodiment, the thermostable ligase isAmpligase®.

4. Ligation Reaction Conditions

Ligation reactions may generally contain about 1 pM to about 500 nM ofeach nucleic acid patch oligo, about 1 pM to about 500 nM of eachuniversal primer, about 3, 4, 5, 6, 7, or 8 units of Ampligase®, and 1times Ampligase Reaction Buffer.

In some embodiments, ligation reactions may be performed by thermalcycling between a high temperature to melt the nucleic acid strands, asequence of 1, 2, 3, 4 or 5 lower temperatures to anneal the nucleicacid patch oligonucleotides to the target nucleic acid, and atemperature compatible with the ligase to ligate the nucleic acidsequence. In a preferred embodiment, ligation reactions may be performedby thermal cycling between a high temperature to melt the nucleic acidstrands, a first lower temperature to anneal the nucleic acid patcholigonucleotides to the target nucleic acid, a second lower temperatureto anneal the universal primers to the nucleic acid patcholigonucleotides, and a temperature compatible with the ligase to ligatethe nucleic acid sequence. In one embodiment, the melting temperaturesmay be about 85, 86, 87, 88, 89, 90, 95, or 100 degrees Celsius. In apreferred embodiment, the melting temperature may be about 90, 91, 92,93, 94, 95, 96, 97 or 98 degrees Celsius. In another embodiment, theNucleic acid patch oligonucleotide annealing temperatures may be about30, 35, 40, 45, 50, 55, 60, 65, 70, 75 degrees Celsius or more. In apreferred embodiment, the nucleic acid patch oligonucleotide annealingtemperatures may be about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, or 72degrees Celsius. In another embodiment, the ligation temperature may beabout 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 degrees Celsius ormore. In a preferred embodiment, the ligation temperature may be about55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 degreesCelsius or more.

In some embodiments, the ligation reactions may be incubated at themelting temperature for about 5 to about 60 seconds. In a preferredembodiment, the ligation reactions may be incubated at the meltingtemperature for about 30 seconds. In some embodiments, the ligationreactions may be incubated at the nucleic acid patch oligonucleotideannealing temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreminutes. In a preferred embodiment, the reactions may be incubated atthe nucleic acid patch oligonucleotide annealing temperature for about 2minutes. In some embodiments, the ligation reactions may be incubated atthe universal primer annealing temperature for about 30 seconds to about5 minutes. In a preferred embodiment, the ligation reactions may beincubated at the universal primer annealing temperature for about 1minute. In some embodiments, the ligation reactions may be incubated atthe ligation temperature for about 30 seconds to about 5 minutes. In apreferred embodiment, the ligation reactions may be incubated at theligation temperature for about 1 minute. In some embodiments, thereactions may be pre-incubated at the melting temperature for about 5,6, 7, 8, 9, 10, 15, 20 or 25 minutes before cycling between the melting,annealing and ligation temperatures. In a preferred embodiment, theligation reactions may be pre-incubated at the melting temperature forabout 15 minutes.

In some embodiments, the ligation reactions may be cycled between themelting, annealing and ligation temperatures about 10, 50, 100, 150, 200or more times. In a preferred embodiment, the ligation reactions may becycled between the melting, annealing and elongation temperatures about100 times.

H) Degrade Mispriming Products and Genomic DNA

In some embodiments of the PDP and PtG methods and processes hereindisclosed, exonucleases may be added to the ligation reaction at thecompletion of the reaction to degrade mispriming products of themultiplex PCR reaction or genomic DNA. In preferred embodiments,exonucleases may be 3′ to 5′ exonucleases. Exonucleases may be singlestranded or double stranded exonucleases. Non-limiting examples ofexonucleases suitable for this step of the reaction may includeexonuclease I, exonuclease III and mung bean nuclease. One or moreexonucleases may be added. In a preferred embodiment, the exonucleasesmay be exonuclease I and III.

I) Sample-Specific Barcode PCR and Sequencing of Resultant Nucleic AcidPatch Amplicons

In some aspects of the invention, nucleic acid samples may be sequenced.In some embodiments, the nucleic acids sequenced may be the ampliconsprepared in PDP and PtG described above. Sequencing techniques suitablefor the invention may be high throughput. High throughput sequencingtechniques may include techniques based on chain termination,pyrosequencing (sequence by synthesis), or sequencing by ligation andare well known to those of skill in the art. In some embodiments, highthroughput sequencing techniques like true single molecule sequencing(tSMS) may not require amplification of target nucleotide sequences. Inpreferred embodiments, sequencing may he performed using high throughputsequencing techniques that involve in vitro clonal amplification of thetarget nucleotide sequence. Non-limiting examples of high throughputsequencing techniques that involve amplification may include solid-phasePCR in polyacrylamide gels, emulsion PCR, rolling-circle amplification,bridge PCR, BEAMing (beads, emulsions, amplification andmagnetics)-based cloning on beads, massively parallel signaturesequencing (MPSS) to generate clonal bead arrays. In a preferredembodiment, the amplicons may be sequenced using PCR techniques asexemplified by 454 Sequencing TM.

In some embodiments, the PCR may use primers complementary to theuniversal primer sequences. In other embodiments, the PCR primers may becoupled to nucleic acid sequences for sequencing. In a preferredembodiment, the primers for the final universal PCR may be tailed to 454sequencing primers A and B (454 Life Sciences, Branford, Conn.). Inother embodiments, the primers for the PCR amplification may becomplementary to upstream and downstream universal primer nucleotidesequences. In additional embodiments, the PCR primers may be coupled tonucleic acid sequence barcodes. In some embodiments, the nucleic acidbarcode may be about 4, 5, 6, 7, 8, 9, 10, or more bases. In a preferredembodiment, the nucleic acid barcode may be about 6 bases. The barcodesmay be at the 5′ end, the 3′ end or, internal to the primer sequence.

In some embodiments, nucleic acid sequences amplified in the PCRreactions of more than one sample may be pooled for parallel sequencingof nucleic acids prepared in multiple samples. In some embodiments,about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 ormore samples may be pooled for sequencing.

While the invention has been disclosed in conjunction with a descriptionof certain embodiments, including those that are currently believed tobe the preferred embodiments, the detailed description is intended to beillustrative and should not be understood to limit the scope of thepresent disclosure. As would be understood by one of ordinary skill inthe art, embodiments other than those described in detail herein areencompassed by the present invention. Modifications and variations ofthe described embodiments may be made without departing from the spiritand scope of the invention.

1. A method for the capture and amplification of a targeted region offragmented deoxyribonucleic acid (fgDNA), the method comprising:providing a sample of an fgDNA having a minimal targeted region betweentwo unknown sequences; reducing said sample of said fgDNA to singlestranded fgDNA; adding oligonucleotide patches to said single strandedfgDNA, said oligonucleotide patches comprising: a nucleotide sequencethat is a reverse compliment to an end sequence that defines the minimaltargeted region; a universal primer sequence; and a protecting group onat least one end; said oligonucleotide patches binding to said endsequence to create: double stranded DNA at said end sequence; a singlestranded DNA flanking region outside said minimal targeted region, saidsingle stranded DNA flanking region being of unknown length andsequence; and a single stranded DNA gap region between said universalprimer sequence and said end sequence; filling in said single strandedDNA gap region with polymerase to make double stranded DNA at said gapregion; adding enzymes to said single stranded fgDNA, said enzymescleaving said single stranded DNA flanking regions from said doublestranded DNA, resulting in said single stranded fgDNA being reduced to aportion of said minimal targeted region with said double stranded DNA atboth ends, said protecting group preventing said oligonucleotide patchfrom degradation by said enzymes; ligating a complementary universalprimer to each of said ends of said targeted region; performingpolymerase chain reaction; and sequencing the polymerase chain reactionamplicons of said targeted region.
 2. The method of claim 1, wherein amultiplicity of oligonucleotide patch pairs are added to the reactionconcurrently.
 3. A system for the capture and amplification of atargeted region of fragmented deoxyribonucleic acid (fgDNA), the systemcomprising: a sample comprising: single stranded fgDNA reduced fromdouble stranded fgDNA, said single stranded fgDNA having a minimaltargeted region between two unknown sequences; and a plurality ofoligonucleotide patches, each of which comprises: a nucleotide sequencethat is a reverse compliment to an end sequence that defines the minimaltargeted region; a universal primer sequence; and a protecting group onat least one end; said oligonucleotide patches binding to said endsequence to create: double stranded DNA at said end sequence; a singlestranded DNA flanking region outside said minimal targeted region, saidsingle stranded DNA flanking region being of unknown length andsequence; and a single stranded DNA gap region between said universalprimer sequence and said end sequence; enzymes for cleaving said singlestranded DNA flanking regions from said double stranded DNA at both saidends of said single stranded fgDNA, resulting in said single strandedfgDNA being reduced to said targeted region with said double strandedDNA at both said ends said protecting group preventing saidoligonucleotide patch from degradation by said enzymes; and acomplementary universal primer having a complementary sequence to saiduniversal primer sequences of said oligonucleotide patches bound to eachof said ends of said targeted region.
 4. The system of claim 3, whereinall said plurality of oligonucleotide patches are bound to said ends.